David B. Weinstein

Effects of Insulin and Glucose on Very Low Density Lipoprotein Triglyceride Secretion by Cultured Rat Hepatocytes

The effect of insulin on hepatic triglyceride synthesis and secretion is controversial. Previously, we have described a cell culture system of adult rat hepatocytes that synthesize and secrete very low density lipoprotein (VLDL) triglycerides with small and irreproducible effects of insulin on triglyceride metabolism. To study the primary effects of insulin on hepatic triglyceride metabolism a method was developed utilizing fibronectin-coated culture dishes that allowed adhesion, spreading, and maintenance of hepatocytes for 2-3 d in the absence of serum and insulin. This culture system allowed mass measurements of both cellular and secreted VLDL triglycerides for long time periods after the addition of physiological concentrations of insulin to hormone-free culture medium. In the absence of insulin and after an initial 4 h in culture, the medium was replenished and triglyceride mass was measured at the end of 18-h incubations. VLDL triglyceride accumulated in the culture medium at a linear rate over this time-course with increasing accumulation as the medium glucose concentration was raised from 2.5 to 25 mM glucose (1.77+/-0.24 to 3.09+/-0.76 mug triglyceride/mg cell protein per h). There was no apparent significant lipolysis or hepatocellular reuptake of secreted VLDL triglycerides. In the absence of insulin cellular triglyceride levels were unchanged between 3 and 24 h in culture while insulin (50-500 muU/ml) significantly increased cellular triglyceride content at all glucose concentrations tested (0-25 mM). The addition of insulin to the culture medium progressively reduced the rate of VLDL triglyceride secretion accompanied by an increase in cellular triglyceride at insulin concentrations > 50 muU/ml. Most or all of the observed increase in cell triglyceride content could in all experiments be accounted for by the insulin-induced inhibition of VLDL secretion. Incorporation of [2-(3)H]glycerol into cellular and VLDL triglycerides as a function of insulin concentration was also measured. Glycerol incorporation data at 20-22 h after plating of the cells closely paralleled the insulin-induced changes in cellular and VLDL triglyceride as determined by mass analysis. The observed effects of insulin occurred at concentrations close to the physiological range and suggest that the direct hepatic effect is to suppress VLDL secretion although the net effect in vivo will clearly reflect many additional accompanying changes.

Interaction between High Density and Low Density Lipoproteins during Uptake and Degradation by Cultured Human Fibroblasts

High density lipoprotein (HDL) inhibited the binding (trypsin-releasable radioactivity), internalization (cell-associated radioactivity after trypsinization), and degradation (TCA-soluble non-iodide radioactivity) of (125)I-low density lipoprotein ((125)I-LDL) by cultured normal human fibroblasts. At HDL:LDL molar ratios of 25:1 (protein ratios about 5:1), these parameters were reduced by about 25%. Unlabeled LDL was about 25 times more effective in reducing (125)I-LDL binding, implying that if HDL and LDL bind at common sites the affinity of HDL for these sites is very low or that the interaction is on some other basis. The fractional reduction in (125)I-LDL binding at a given HDL: (125)I-LDL ratio was independent of (125)I-LDL concentration and occurred equally with fibroblasts from a subject with homozygous familial hypercholesterolemia. Reciprocally, the binding, internalization, and degradation of (125)I-HDL were reduced by LDL. Preincubation of fibroblasts with HDL (or LDL) reduced the subsequent binding of (125)I-LDL (or (125)I-HDL) during a second incubation. In other studies HDL reduced the net increase in cell cholesterol content induced by incubation with LDL. HDL alone had no net effect on cell cholesterol content. These findings suggest that HDL reduces both the high affinity and the low affinity binding of LDL to human fibroblasts and that this in turn reduces the internalization and degradation of LDL. The effect of HDL on the LDL-induced changes in cell cholesterol content could be in part on this basis and in part on the basis of an HDL-stimulated release of cholesterol from the cells. These effects of HDL in vitro may be relevant to the negative correlations reported from in vivo studies between plasma HDL concentration and both body cholesterol pool size and the prevalence of clinically manifest atherosclerosis but further studies will be needed to establish this.

A Novel Role for the Immunophilin FKBP52 in Copper Transport

FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes. Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood. To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library. We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone. Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux. The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506. Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper. Overexpression of FKBP52 increased rapid copper efflux in 64Cu-loaded cells, as did the overexpression of WD transporter. Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity.

The Role of the Liver in LDL Catabolism

In 1974 we showed that after total hepatectomy both pigs and dogs degrade injected 125I-LDL at a rate equal to or actually greater than the rate in the intact animal (Sniderman et al. 1974). Those results established for the first time the large potential capacity of extrahepatic tissues to degrade LDL in vivo. However, no final quantitative conclusions could be reached regarding the relative roles of liver and extrahepatic tissues in the intact animal because of the possible perturbations accompanying hepatectomy. The very fact that fractional catabolic rate increased after hepatectomy indicated that some acute change induced by the procedure must influence extrahepatic LDL metabolism. A hypothesis that might explain the phenomenon was advanced (Steinberg et al. 1974) but others are possible and the basis for the paradoxical finding remains undefined. Nor did those studies provide any independent assessment of the relative contribution of different extrahepatic tissues to overall LDL degradation.

Synthesis and Secretion of Very Low Density Lipoprotein by Cultured Rat Hepatocytes

Cells of many animal species and of diverse tissue origins can be grown and maintained under defined and reproducible conditions in cell culture. During the past five years we have gained information and valuable insight from cell culture studies of lipid synthesis, lipoprotein uptake and degradation and factors which regulate the net cholesterol content of cells and tissues. Recent studies on the hepatic contribution to lipoprotein synthesis have utilized either freshly isolated rat hepatocytes (Jeejeebhoy and Phillips 197 6) or single-pass and recirculating rat liver perfusions (Marsh 1974, 1976; Hamilton et al. 1976). Primary monolayer cultures of adult rat hepatocytes which maintain hepatic biosynthetic functions for periods of several days, rather than hours, can provide advantages over the short-term isolated cell and organ perfusion systems.